The sequencing of the human genome presents enormous opportunities for the determination of the mechanisms of many protein-based diseases. However, the interpretation and application of acquired genetic data will rely, at least in part, on an ability to determine protein secondary, tertiary and quaternary structure. Although the Human Genome Project can potentially unlock the amino acid sequence of many proteins, the activity of a protein is not determined only its amino acid sequence. Higher order structure plays an equally important, if not more important, role. Indeed, the prediction of secondary structure based on primary sequence is one of the most important problems in biology.
A goal of protein folding research efforts has been to solve various forms of protein structure by analyzing the primary amino acid sequence and thus to allow an interpretation of human genome sequence data in terms of protein structure. Teichmann et al., (2000) Bioinformatics 16:117-24; Zhang & Zhang, (2000) Biopolymers 53: 539-49. This effort, however, is far from complete and currently available technology is expected to rely heavily on the field of proteomics to determine the structure and function of the vast number of proteins now known only by their primary amino acid sequence. Preferably, techniques that provide rapid access to secondary structure information can be combined with known primary sequence information and homology modeling to gain structural information on a large number of proteins. Teichmann et al., (2000) Bioinformatics 16:117-24.; Zhang & Zhang, (2000) Biopolymers 53: 539-49.
Infrared (IR) spectroscopy is well established as a valuable technique for assessing protein secondary structure in solution. One particular form of IR spectroscopy, Fourier transform infrared spectroscopy (FTIR), has become an especially preferred form of IR spectroscopy for the study of protein secondary structure. FTIR has great utility in the rapid determination of secondary structure because it offers accurate, high-resolution spectra with excellent sensitivity and signal-to-noise (S/N) ratios, as compared to other forms of infrared spectroscopy. Kumosinski & Unruh, (1994) in ACS Symposium Series 576, Molecular Modeling: From Virtual Tools to Real Problems, (Kumosinski & Liebman, eds.) pp. 71-98; Susi & Byler, (1986) Method. Enzymol. 130: 290-311. Over the last thirty years, these properties of FTIR have been increasingly recognized and FTIR has developed into a reliable and accurate technique for the identification of structural features of a variety of sample, including protein secondary structure. Susi & Byler, (1986) Method. Enzymol. 130: 290-311; Byler & Susi (1986) Biopolymers 25: 469-87; Jencks, (1986) Method. Enzymol. 6 (125): 914-29; Douseeau & Pezolet, (1990) Biochem. 29: 8771-79; Purcell & Susi, (1984) J. Biochem. Bioph. Meth. 9: 193-99; Miyazawa, (1960) J. Chem. Phys. 32(6): 1647-52; Krimm, (1962) J. Mol. Biol. 4: 528-40; Krimm & Abe, (1972) Proc. Nat. Acad. Sci. 69 (10): 2788-92; Miyazawa et al., (1956) J. Chem. Phys. 24(2): 408-18.
Proteins are known to have nine characteristic absorption bands in the mid-infrared region (approximately 1250 cm−1 to 1850 cm−1) that yield conformational insight and are known as the amide A, B, and I-VII bands. Susi & Byler, (1986) Method. Enzymol. 130: 290-311; Susi (1972) Method. Enzymol. 26 Pt.C: 455-72. The secondary structure of proteins has primarily been characterized by the frequency of the amide I and II bands. Susi & Byler, (1986) Method. Enzymol. 130: 290-311; Jencks, (1986) Method. Enzymol. 6 (125): 914-29; Miyazawa (1960) J. Chem. Phys. 32(6): 1647-52; Krimm (1962) J. Mol. Biol. 4: 528-40; Krimm & Abe, (1972) Proc. Nat. Acad. Sci. 69(10): 2788-92; Miyazawa et al., (1956) J. Chem. Phys. 24(2): 408-18; Susi, (1972) Method. Enzymol. 26 Pt. C: 455-72. The characterization of protein samples almost exclusively by the amide I and amide II bands of the protein's IR spectrum, is due to limitations imposed by the presence of large solvent bands of both water (H2O) and deuterium oxide (D2O) that obscure regions of the infrared spectrum where additional informative bands can be observed. In effect, the solvent bands overlap the other bands present in a sample, which, therefore, cannot be clearly observed.
There is, however, a volume of information that can be used to derive structural information about a sample by analyzing the shape and position of bands in the amide I region of the spectrum. Studies have indicated that the quantity and quality of various amide I band frequencies are indicative of the presence of α-helices, β-sheets and random coil structures. Yang et al., (1985) Appl. Spectrosc. 39(2): 282-87; Byler & Susi, (1986) Biopolymers 25: 469-87; Susi & Byler, (1988) Appl. Spectrosc. 42(5): 819-25; Matsui & Tanaka, (1987) Appl. Spectrosc. 41(5): 861-65; Jakobsen et al., (1986) Biopolymers 25: 639-54; Wasacz et al., (1987) Biochem. 26: 1464-70. Often, however, conventional IR techniques cannot identify this information due to overlap of solvent signals with protein signals.
Attenuated total reflectance (ATR) is a technique useful for spectrally analyzing liquids having absorptions that are too strong for conventional transmission analysis. This condition is commonly encountered in the infrared (IR) region of the spectrum, which is the spectral region that encompasses the fundamental frequencies of most molecular vibrations. ATR has also found some application in the ultra-violet (UV) and visible regions for the analysis of dyes and other strongly absorbing water-soluble substances.
Water is an ideal solvent for biological samples. However, when used in transmission FTIR experiments it causes serious errors in the amide I region (1630-1690 cm−1) due to the strong absorption of H2O at about 1640 cm−1. Susi & Byler, (1986) Method. Enzymol. 130: 290-311; Byler & Susi, (1986) Biopolymers 25: 469-87; Jencks, (1986) Method. Enzymol. 6 (125): 914-29; Douseeau & Pezolet, (1990) Biochem. 29: 8771-79; Susi, (1972) Method. Enzymol. 26 Pt.C: 455-72. The absorption of water masks the absorption of a protein to such an extent that cell pathlengths of less than 6 microns must be used when analyzing a protein in an aqueous solution. Douseeau & Pezolet, (1990) Biochem. 29: 8771-79; Chittur, (1998) Biomaterials 19: 357-69. Furthermore, only the amide II region (1480-1575 cm−1) can be reliably obtained in most protein spectra in which the protein is disposed in an aqueous solution. Polytetrafluoroethylene and other spacers are not generally available in thicknesses of less than 6 microns, thus limiting options for transmission FTIR experiments in aqueous solution. Because H2O signals can mask the amide I region, deuterium oxide (D2O) is often used as a solvent. The strong D-O-D and H—O—H bending modes, however, still obscure the spectral regions from 1150-1250 cm−1 and 1600-1800 cm−1, respectively. Jencks, (1986) Method. Enzymol. 6 (125): 914-29.
In theory, both the amide I and amide II protein bands can be resolved, if spectra in both H2O and D2O solvents are obtained. The deuterium exchanged amide I and amide II bands are referred to as amide I′ and amide II′ bands in the spectroscopy literature. However, the exchange of proteins in D2O for the determination of spectra is tedious and can compromise the integrity of the sample. There are at least two notable problems associated with the use of D2O: first, hydrogen atoms within the protein exchange with deuterium over wide range of time scales unless the protein is fully denatured (Susi, (1972) Method. Enzymol. 26 Pt.C: 455-72; Powell et al., (1986) Appl. Spectrosc. 40(3): 339-44; Nabet & Pezolet, (1997) Appl. Spectrosc. 51(4): 466-69; Dousseau et al., (1989) Appl. Spectrosc. 43(3): 538-42); and second, bands due the species H—O-D, D-O-D, and H—O—H are all present upon the introduction of D2O unless an absolutely complete exchange of hydrogen for deuterium, a process known as “deuteration”, is achieved. Jencks, (1986) Method. Enzymol. 6 (125): 914-29. The exchange of N—H with N-D also shifts the amide II′ band from 1550 to 1450 cm−1 (Jencks, (1986) Method. Enzymol. 6 (125): 914-29; Dousseau et al., (1989) Appl. Spectrosc. 43(3): 538-42) and the presence of H—O-D in solution overlaps the amide II′ and amide A′ regions.
For reasons of convenience, the majority of FTIR structural investigations have focused on secondary structure information acquired from protein spectra of the amide I′ band in D2O solution. The current state of the art for FTIR investigation of secondary structure requires placing proteins between two salt windows (typically these windows are fashioned of calcium fluoride or other salt) and orienting the windows so that they are separated by a thin pathlength space. An IR spectrum is subsequently acquired. Typical pathlength spaces are on the order of 6 to 25 microns. Barium fluoride, zinc selenide and other materials are also used for manufacturing windows.
These windows, however, can be costly and fragile and can have short lifespans. Additionally, such cells have the associated drawbacks that it is difficult to inject a sample into the ultrathin pathlength and to extract a sample from the ultrathin pathlength. Sample recovery can be a primary concern when the sample comprises a quantity of protein that is difficult to purify. A viable alternative to the traditional IR techniques is the use of the FTIR-ATR techniques. However, even certain FTIR-ATR techniques have drawbacks when the technique is used to study protein in aqueous solution. For the bending mode of water, there is typically an absorbance of 0.04 per reflection. Thus, for a typical waveguide with about 20 reflections, the absorbance of water is about 0.8 in the region of the amide I band. Solvent subtraction is possible but tedious in this case. In the region of the amide A band, the absorbance of the symmetric OH stretch is 0.16 per reflection. Thus, for a typical waveguide, the absorbance is about 3.2, prohibiting observation in this region. Multi-pass FTIR-ATR techniques that can be useful to acquire a spectrum from non-aqueous samples cannot, therefore, be effectively applied to protein samples which are best disposed in aqueous buffer to maintain sample integrity.
Thus, what is needed is a method that permits the observation of the spectral bands of a sample that appear in the IR region of the spectrum. A desirable method would be inexpensive, accurate and would eliminate the need for specialized equipment. Such a method would be easily automated for rapid data acquisition and analysis of many samples.